Project Summary Auditory hair cells are mechanotransduction receptors that convey sound signals to the brainstem via spiral ganglion neurons (SGNs). However, hair cells are vulnerable to a number of insults, including noise, blast, aging, and traumatic brain injury. Adult mammalian hair cell loss is usually irreversible, causing permanent hearing loss and other inner ear disorders. Currently, there are no biological approaches to replenish human hair cells. The long-term goal of this project is to use stem cell-based approaches to regenerate the damaged auditory system to treat hearing loss and other inner ear disorders. In this proposal, we will generate human sensory hair cells and rebuild hair cell-SGN connections using a stem cell-based epigenetic approach. We have identified human utricle epithelia-derived prosensory-like cells (HUCs) that exhibit features of prosensory cells. We found that HUCs demonstrated genomic DNA methylation in a pilot study. We applied the DNA methyltransferase (DNMT) inhibitor 5-azacytidine (5-aza) to HUCs and found that treated HUCs expressed hair cell genes and proteins. Therefore, in this proposal we hypothesize that the DNMT inhibitor 5- aza induces HUCs to differentiate into new functional hair cells that can replace damaged hair cells and form synapses with auditory neurons. To test this hypothesis, three specific aims are proposed: Specific Aim 1: Define the induction of HUCs into sensory hair cells by 5-aza. Specific Aim 2: Investigate the integration and synapse formation of 5-aza-treated HUCs in vitro. Specific Aim 3: Determine the survival, differentiation, integration, and function of 5-aza-treated HUCs in vivo. In this proposal, we will generate human sensory hair cells and rebuild hair cell-neuron connections using a stem cell-based epigenetic approach. The advantage of an epigenetic approach is that expression and phenotype of gene are heritably changed, whereas their DNA sequences remain constant. The long-term aims of this work are (a) to discover and explore innovative regeneration approaches to improve the health and welfare of military personnel and veterans with hearing disturbances, and (b) to accelerate the transition of stem cell technologies into new standards of care to treat hearing loss and other inner ear disorders. Success of this work will significantly promote the research of stem cell-based hearing regeneration, which will potentially affect the state of medical science in today?s battlefield experience on function, wellness, and overall quality of life for veterans as well as their caregivers and families.